Modulation of the Transcription of Pro-Inflammatory Gene Products

ABSTRACT

The present invention refers to inhibitors of the transcription factors IRF-1, their use as therapeutic agents as well as their use for prevention and therapy of cardiovascular complications like re-stenosis after percutaneous angioplasty or stenosis of venous bypasses, chronic (transplant arteriosclerosis or vasculopathy) or acute transplant rejection, graft versus host disease (GVHD), immunological hypersensitivity reactions (allergies), particularly bronchial asthma and atopic dermatitis, chronic recurrent inflammatory diseases, particularly ulcerative colitis and Crohn&#39;s disease, psoriasis and sarcoidosis, as well as autoimmune diseases, particularly diabetes mellitus, multiple sclerosis, collagenoses (e.g. systemic lupus erythematodes), rheumatoid arthritis and vasculotids.

The present invention refers to inhibitors of the transcription factorsIRF-1, their use as therapeutic agents as well as their use forprevention and therapy of cardiovascular complications like re-stenosisafter percutaneous angioplasty or stenosis of venous bypasses, chronic(transplant arteriosclerosis or vasculopathy) or acute transplantrejection, graft versus host disease (GVHD), immunologicalhypersensitivity reactions (allergies), particularly bronchial asthmaand atopic dermatitis, chronic recurrent inflammatory diseases,particularly ulcerative colitis and Crohn's disease, psoriasis andsarcoidosis, as well as autoimmune diseases, particularly diabetesmellitus, multiple sclerosis, collagenoses (e.g. systemic lupuserythematodes), rheumatoid arthritis and vasculotids.

The endothelium of blood vessels plays a key role in inflammatorydiseases because it represents the primary interaction site forcirculating inflammation competent cells with the tissue. In acute orchronic inflammation manifold interactions between endothelium cells andboth monocytes and polymorphonuclear neutrophil granulocytes aredescribed. Recently the interaction between endothelium cells andpro-inflammatory T helper cells (TH1) in autoimmune diseases (e.g.rheumatoid arthritis), arteriosclerotic lesions of blood vessels wallsincluding transplant and venous bypass vasculopathy as well asre-stenosis after percutaneous angioplasty and in chronic recurrentinflammatory diseases (e.g. Crohn's disease, psoriasis) are increasinglydiscussed. Lymphocytes and endothelium cells communicate over theCD40/CD154 receptor/ligand system (also known as TNFreceptor/ligand-5-system) with consecutive increase of expression ofchemokine and adhesion molecules in the endothelium. Moreover,endothelium cells in contrast to other antigen presenting cells likemonocytes seem to release biological active interleukin 12 solely afteractivation of the CD40 signalling pathway in an amount similar tomaximally stimulated monocytes (these are generally thought to be themain source of interleukin 12). Interleukin 12 is the primary stimulusand differentiation factor, respectively, for naive T helper cells whichreact with an increased production of interferon γ and expression ofCD154, respectively, on their surface (these T helper cells are thenregarded as TH1 cells). Interferon γ in turn increases the expression ofCD40 in endothelium cells resulting in a vicious cycle in whichendothelium cells, T-helper cells and recruited monocytes stimulate eachother and keep the inflammatory reaction going.

The co-stimulating properties of CD40/CD154 which trigger theinflammation have been demonstrated in animal models for diseasesincluding Crohn's disease and acute or chronic transplant rejection(vasculopathy). Not only the endothelium leukocyte interaction viaCD40/CD154 plays a role here, but also for example theCD40/CD154-mediated interaction of monocytes/macrophages or dendriticcells with TH1-cells and naive T helper cells, respectively. FurtherCD40 may e.g. be expressed by smooth muscle cells in the vessel liningand also by keratinocytes in skin or synovial fibroblasts in joints.Activation of the CD40 pathway in these cells is furthermore not only ofimportance for inflammatory reactions, but also leads to rebuildingprocesses in tissue as for example remodelling of vessel lining intransplant vasculopathy, skin changes in psoriasis or erosions of jointcartilage in rheumatoid arthritis. Beside CD154 induced, interleukin 12depend and TH1 mediated chronic inflammatory diseases and autoimmunereactions, respectively, including Diabetes mellitus, multiplesclerosis, sarcoidosis and vasculotids the co-stimulatory properties ofCD40/CD154 are also important for differentiation of B-lymphocytes inantibody producing plasma cells which is triggered by contact withTH2-cells. Thereby B-lymphocytes express CD40 and TH2-cells expressCD154. Without this co-stimulation plasma cells produce primarilyantibodies of the IgM type and barely antibodies of type IgE or IgG. Anexaggerated TH2 response, i.e. excessive production of type IgE or IgGantibodies plays an important role in mainly allergy caused chronicrecurrent inflammatory diseases as bronchial asthma, atopic dermatitisand ulcerative colitis but also in collagenoses as systemic lupuserythematodes (SLE), in which the production of autoreactiveautoantibodies is of special importance and which is therefore regardedas a generalized autoimmune disease. In general differentiation betweenautoimmune diseases and chronic recurrent inflammatory diseases isproblematic because a common predisposing factor seems to be theimbalance between TH1 and TH2 mediated cellular and humoral immunereaction, respectively.

Presently the only useful therapeutic approach for the treatment ofdiseases inter alia associated with the CD40/CD154 signallingpathway—apart from blocking antibodies against CD 154—is the inhibitionof CD40 expression in CD154 target cells. One of the drawbacks of thetreatment with anti-CD154-antibodies is the risk of hypersensitivityreactions (against the antibody), particularly with repeatedapplication, and the poor accessibility of at least tissue-basedepitopes (e.g. infiltrated T-lymphocytes) because antibodies must beapplied into the blood. However, as for many other cytokine receptors,too, there are no small molecular receptor antagonists for CD40. Due totrimerization of the receptor molecules after ligand binding CD40antibodies tend to activate CD154 target cells. Other strategiesdelimiting from the commonly decay of the inflammatory reactions consistof the stimulation of the TH1 cell response at the preponderance of theTH2 cell response (e.g. by administering of a TH1 cytokine likeinterferon γ) or vice versa by stimulation of the TH2 cell response atthe preponderance of the TH1 cell response (e.g. by administering of aTH2 cytokine like interleukin-10). Because the T helper cell reactionscancel out each other by means of cytokine mediation (i.e. thepreponderance of the TH1 cell response leads to a decay of the TH2 cellresponse and vice versa) these strategies hold the danger to disinhibitthe respective other pathway of the T helper cell response. This may inturn leads to the possibility of the respective other inflammatoryreaction.

Thus, one problem of the present invention is the provision of agentsfor prevention and/or therapy of inflammation diseases, which amongothers are associated with the CD40/CD154 co-stimulation.

The problem is solved by the subject matter defined in the claims.

The invention is illustrated by the following figures.

FIG. 1 shows graphically the result of the CD40 mRNA expression (RT-PCRanalysis) in not-stimulated TNFα (1000 U/ml), IFNγ (1000 U/ml) and TNFα(100 U/ml) plus IFNγ (1000 U/ml)-stimulated cultivated human endotheliumcells after 9 hours (% related to the basal CD40 expression innot-stimulated endothelium cells) (n=5-9, *P<0.05 versus basal, †P<0.05versus TNFα and IFNγ).

FIG. 2 shows schematically the result of the time dependent increase ofthe nuclear translocation of NFκB (p65/p50 heterodimer) of the p91/p91homodimers of STAT-1 and of IRF-1 in human endothelium cells, incubatedfor 0.5 hours (NFκB and STAT-1) and. 3 hours (IRF-1) with TNFα (1000U/ml), respectively, IFNγ (1000 U/ml) and TNFα (100 U/ml) plus IFNγ(1000 U/ml). A pre-incubation (1 hour) with cycloheximide (Cx, 1 μM)demonstrates, that IRF-1 is expressed de novo. Representativeelectrophoretic mobility shift analysis, comparable results wereobtained in further experiments.

FIG. 3 shows schematically the results of specific effects ofCis-element decoys against STAT-1 NFκB and IRF-1 (10 μM, 4 hpre-incubation) on (a) the mRNA level of CD40 (n=3-5, statisticalsummary, in % related to the maximum value, *P<0.05 versus TNFα/IFNγ),(b) the mRNA-level of CD40 and E-selectine (representativeRT-PCR-analysis, comparable results were obtained in furtherexperiments), (c) the CD40 protein level (representative western blot,comparable results were obtained in further experiments, in humanendothelium cells which were incubated for 9 hours (RT-PCR analysis) and24 hours (western blot), respectively, with TNFα (100 U/ml)/IFNγ (1000U/ml). In the experiments shown under (b) and (c) the relativeintensities (%) determined by densitometrical analysis (One-Dscan-GelAnalysis Software, Scanalytics, Billerica, Mass., USA) are shown inrelation to the maximum values at cytokine stimulation.

FIG. 4 shows schematically the effects of different Cis-element decoysagainst STAT-1, NFκB and IRF-1 (10 μM, 4 h pre-incubation) on the CD40protein level (a) determined via Fluorescence Activated Cell Sorting(FACS) in human endothelium cells which were incubated for 24 hours withTNFα (100 U/ml)/IFNγ (1000 U/ml) and (b) the assay for the cell surfaceprotein PECAM-1 characteristic for endothelial cells. An overlay of theoriginal measurement of the IgG isotypecontrol and from TNFα/IFNγtreated (CD40) and non stimulated (PECAM-1) cells, respectively is shownin (a) and (b) each as well as the logarithmic values of the respectiveaverage fluorescence intensities in a table. Representative experiment,comparable results were obtained in further experiments.

FIG. 5 shows schematically the results of the effects of TNFα (2000U/ml, IFNγ (1000 U/ml) and TNFα/100 U/ml) plus IFNγ (1000 U/ml) on theCD40 and IRF-1 mRNA level, respectively, in human endothelium cellsafter 9 hours incubation. Representative experiment, comparable resultswere obtained in further experiments.

FIG. 6 shows schematically the results of the time dependant increase ofthe CD40 and IRF-1 mRNA expression, respectively, in human endotheliumcells, which were incubated for 0, 0.5, 1.5, 3 and 9 hours with IFNγ(1000 U/ml). Representative experiment, comparable results were obtainedin further experiments.

FIG. 7 shows schematically the specificity of the Cis-element decoyeffect on the CD40 mRNA expression in human endothelium cells. Thepre-incubation (4 hours) with the Cis-element decoy (IRF-1n, cons, 10μM), but not the pre-incubation with the respective mutated controloligonucleotide (IRF-1n mut, 10 μM) inhibits the CD40 mRNA expression incells which were subsequently incubated with TNFα (100 U/ml) and IFNγ(1000 U/ml) for 9 hours. Representative RT-PCR analysis, comparableresults were obtained in further experiments.

FIG. 8 shows the inhibition of the cytokine induced (100 U/ml) TNFα,(1000 U/ml) IFNγ expression of the IRF-1 protein (after 3 hours) and theCD40 mRNA (after 9 hours) in human endothelium cells which were treatedprior for 5 hours with an IRF-1 antisense oligonucleotide (AS; SEQ IDNO:23) (concentration 0.2 μM). The left part of (a) and (b) shows thestatistical summary of three experiments with different cell charges,the each right part a representative western blot and RT-PCR analysis,respectively, and in (b) in addition, the densitometrical interpretation(“intensity”) given in % of the stimulated control and related to theinternal standard β-actin (*P<0.05 compared with the stimulated controlcells). The respective missense (MS) and scrambled (SCR), controloligonucleotides respectively influenced neither the expression of RF-1nor the expression of CD40.

FIG. 9 shows the electrophoretic mobility shift analysis of the uptakeof different IRF-1 Cis-element decoys (SEQ ID NO:13, 17, 19 and 21) incultivated THP-1 cells and the subsequent neutralization of IRF-1. TheTHP-1 cells were pre-incubated with the different Cis-element decoys for1 hour and stimulated subsequently for further 3 hours with TNFα (100U/ml) and IFNγ (1000 U/ml). The result of the following processing andanalysis of the samples is shown in the left part of the image. Theright part of the image shows the electrophoretic mobility shiftanalysis of a nuclear extract of stimulated control cells prepared underidentical experimental conditions. The nuclear extract was incubatedadditionally with an anti IRF-1 antibody as described in Krzesz et al.(1999) FEBS Lett. 453, 191 prior to the electrophoretic mobility shiftanalysis (supershift analysis).

The term “decoy-ODN” or “Cis-element decoy” or “double stranded DNAoligonucleotide” as used herein designates a double stranded DNAmolecule, having a sequence corresponding or being similar to the IRF-1core binding sequence naturally occurring in the genome and to which thetranscription factor IRF-1 binds to said sequence in the cell. TheCis-element decoy thus effects as a molecule for the competitiveinhibition of IRF-1.

The inventors could solve the transcription factors involved in theinflammation dependent, cytokine mediated increase of the CD40 receptorexpression in human endothelium cells. Surprisingly it turned out thatthe transcription factors Nuclear Factor KB (NFκB) and Signal Transducerand Activator of Transcription-1 (STAT-1) control the tumornecrosisfactor α (TNFα)/Interferon-γ (IFNγ)-mediated CD40 expression not in adirect way like in smooth vessel muscle cells of rodents, but in anindirect way by activating of a further transcription factor, namely theInterferon Regulatory Factor-1 (IRF-1). IRF-1 (GenBank Accession No.:L05078, X14454, NM002198 anhttp://transfac.gbf.de/cgi-bin/qt/getEntry.pl?t00423) is a transcriptionfactor being not latent present in the cell in contrast to many othertranscription factors but needs to be synthesized de novo in factnormally after exposition with Interferon-γ and activation of thetranscription factor STAT-1.

In addition Interferon-γ stimulates alone or in combination withtumornecrosis factor α in human endothelium cells the expression ofCD40. In this contents the TNF-α dependent activation of NFκB plays aminor role. More important is the IFN-γ dependent activation of STAT-1leading to the de novo expression of IRF-1. IRF-1 then induces theexpression of CD40. The synergism of the two cytokines is basedessentially on an amplification of the IRF-1 expression. When using thedecoy oligonucleotides against STAT-1 and IRF-1 according to the presentinvention but not the respective control oligonucleotides in human cellsin cell culture the cytokine induced CD40 expression (both inmonostimulation with IRF-γ and in combination of IFN-γ and TNFα) isinhibited. Thereby the induction of IRF-1 precedes the induction ofCD40, so that an antisense oligonucleotide blockade of the IRF-1expression inhibits the cytokine induced CD40 expression in the sameamount as the decoy oligonucleotides. A deactivation of the IRF-1activity in cells results in a high significant and selective inhibitionof the CD40 expression in these cells. As a result of the reduced CD40expression under pro-inflammatory conditions the endothelium leukocyteinteraction particularly the interaction of TH1 and endothelium cellswill be toned down and represents the basis for the therapeuticalsuccess. The same applies in analogy to reduction of the CD40/CD154mediated interaction of naive T helper cells with antigen presentingcells (e.g. monocytes, dendritic cells), of TH2 cells with B lymphocytesas well as other CD40 expressing cells (e.g. smooth muscle cells,ceratinocytes, fibroblasts) with CD154 expressing cells (TH1 cells,activated thrombocytes).

One aspect of the present invention refers therefore to the provision ofan inhibitor of the activity of the transcription factor IRF-1 as atherapeutic substance. Proteins including also IRF-1 may be inhibited indifferent ways in their activity. For example anti IRF-1 antibodies,natural or synthetic substances which reduce the IRF-1 interaction withthe DNA, i.e. the transactivating activity, may be used. The de novosynthesis of IRF-1 may be further inhibited by blockade of STAT-1 andthe signaling path ways (Janus Kinasen) leading to the STAT-1activating, respectively.

A preferred method to specifically inhibit the IRF-1 activity is the useof double stranded DNA oligonucleotides also named Cis-element decoy orDecoy-ODN having a binding site for IRF-1. The exogenous administrationof a large amount of transcription factor binding sites to a cellparticularly in a much higher amount as present in the genome leads to asituation in which the majority of a particular transcription factorbinds specifically to the respective Cis-element decoy but not to theendogenous target binding sites. This approach to inhibit the binding oftranscription factors to their endogenous binding site is also named“squelching”. Squelching (also named neutralization) of transcription byuse of Cis-element decoys was successfully employed to inhibit thegrowth of cells. DNA fragments were used thereby comprising the specifictranscription factor binding sites of cell transcription factor E2F(Morishita et al., PNAS, (1995) 92, 5855).

The sequence of a nucleic acid used to prevent the binding of thetranscription factor IRF-1 is for example a sequence to which IRF-1binds naturally in the cell. IRF-1 binds specifically to the motive withthe sequence 5′-SAAAAGYGAAACC-3′, whereby S=C or G and Y=C or T. Thebinding of IRF-1 depends on the repetitive G/CAAA sequences and thedistance between these motives being particularly three nucleotides.Therefore, the Cis-element decoy of the present invention may have thefollowing 13-mer consensus core binding sequence: 5′-SAAAnnnSAAAyy-3′(SEQ ID NO:1), whereby S=C or G, n=A, T, C or G and y=C or T. TheCis-element decoy may further be longer than the 13-mer core bindingsequence and may be elongated at the 5′- and/or 3′-terminus. Respectivemutations in the core binding sequence result in a loss of the bindingof STAT-1 to the decoy oligonucleotide.

As the Cis-element decoy is a double stranded nucleic acid the DNAoligonucleotide according to the present invention comprises not onlythe sense or the forward sequence, but also the complementary antisenseor reverse sequence. Preferred DNA oligonucleotides according to thepresent invention comprise the following 13-mer core binding sequencesfor IRF-1:

5′-CAAAAGCGAAACC-3′, (SEQ ID NO:3) 5′-GAAAAGCGAAACC-3′, (SEQ ID NO:5)5′-CAAAAGTGAAACC-3′, (SEQ ID NO:7) 5′-GAAAAGTGAAACC-3′, (SEQ ID NO:9)whereby the respective complementary sequences are not shown. However,the Cis-element decoy may comprise a different sequence to the sequencesdescribed above and may be longer than a 13-mer.

The following sequences are more preferred:

(SEQ ID NO:11): 5′-CAGAAAAGTGAAACCCTG-3′, 18-mer (not palindromic, 1binding site), (SEQ ID NO:13): 5′-CAGTTTCAAATTGAAACTG-3′, 19-mer (almostpalindromic, 2 binding sites), (SEQ ID NO:15):5′-CAGGAAAAGTGAAACCGCTG-3′, 20-mer (not palindromic, 1 binding site),(SEQ ID NO:17): 5′-GCAGTTTCAAATTGAAACTGC-3′, 21-mer (almost palindromic,2 binding sites), (SEQ ID NO:19): 5′-GGAAGCGAAAATGAAATTGACT-3′, 22-mer(primary used consensus-sequence), (SEQ ID NO:21):5′-GGCAGTTTCAAATTGAAACTGCC-3′, 23-mer (almost palindromic, 2 bindingsites).

The wording “2 binding sites” refers to the sense and antisense strand.The listing of the preferred sequences is not limiting. It is obviousfor the skilled person that a multitude of sequences may be used asinhibitors for IRF-1 as long as they comprise the conditions listedbefore of the 13-mer consensus core binding sequence and an affinity toIRF-1.

The affinity of the binding of a nucleic acid sequence to IRF-1 may bedetermined by the electrophoretic mobility shift assay (EMSA) (Sambrooket al (1989) Molecular Cloning. Cold Spring Harbor Laboratory Press;Krzesz et al. (1999) FEBS Lett. 453, 191). This assay system is suitablefor the quality control of nucleic acids which are being used for themethod of the present invention or is suitable to determine the optimallength of a binding site. The assay system is also suitable for theidentification of further sequences which are being bound by IRF-1. Mostsuitable for an EMSA which should be used for the isolation of newbinding sites are purified or recombinant expressed versions of IRF-1which are used in several alternating rounds of PCR multiplications andselection by EMSA (Thiesen and Bach (1990) Nucleic Acids Res. 18, 3203).

Genes being known to include IRF-1 binding sites in their promoter orenhancer regions and being therefore putative targets for the specificsquelching by the method of the present invention are for example theCD40 gene and further pro-inflammatory genes e.g. cyclooxygenase-2,subunits of the NADPH oxidase (p67phox and gp91phox), the inducibleisoform of the nitrogen monoxide (NO)-synthase, the interleukins 6, 8and 12 as well as the adhesion molecules RANTES (secreted from Tlymphocytes in soluble form, regulated upon activation, normal T cellexpressed, presumed secreted) and VCAM-1 (vascular cell adhesionmolecule-1, named also CD106).

The method of the present invention modulates the transcription of oneor more genes in such a way that the gene or the genes, e.g. CD40, arenot at all expressed or expressed in a reduced manner. Reduced orinhibited expression in the sense of the present invention means thatthe transcription rate is reduced in comparison to cells being nottreated with the double stranded DNA oligonucleotide according to thepresent invention. Such a reduction may be determined e.g. by NorthernBlotting (Sambrook et al., 1989) or RT-PCR analysis (Sambrook at al.,1989). Such a reduction is typically at least a twofold, preferably atleast a fivefold, more preferably at least a tenfold reduction. The lossof activation may be achieved for example when IRF-1 acts as atranscription activator on a certain gene and therefore, this squelchingof the activator leads to the loss of expression of the target gene.

In addition the method of the present invention enables thedisinhibition of the expression of a gene provided that the expressionis blocked by a constitutively active or (after correspondingstimulation of the cell) an activated transcription factor. One exampleis the disinhibition of the expression of the Prepro-endothelin-1 genein native rabbit endothelium cells of V. jugularis by a Cis-elementdecoy against the transcription factor CCAAT/enhancer binding protein(Lauth et al., J. Mol. Med., (2000), 78, 441). The expression of genesthe products of which exhibit a protective effect for example againstinflammatory diseases may be disinhibited in this way.

The Cis-element decoy used in the present invention includes in apreferred embodiment one or more, preferably 1, 2, 3, 4 or 5, morepreferred 1 or 2 binding sites to which IRF-1 specifically binds. Thenucleic acids may be generated in synthetically, with enzymatic methodsor in cells. The respective methods are state of the art and known tothe skilled person.

The length of the double stranded DNA oligonucleotide is at least aslong as the used sequence binding specifically to IRF-1. Usually theused double stranded DNA oligonucleotide is between about 13-65 bp,preferably between about 13-26 bp and most preferred between 18-23 bplong.

Usually oligonucleotides are degraded rapidly by endonucleases andexonucleases in particular DNases and RNases in the cell. Therefore, DNAoligonucleotides may be modified to stabilize them against degradationso that a high concentration of the oligonucleotides is maintained inthe cell for a longer time. Typically such a stabilization may beachieved by the introduction of one or more modified internucleotidelinkages.

A successfully stabilized DNA oligonucleotide contains not necessarily amodification at each internucleotide linkage. Preferably theinternucleotide linkages are modified at the respective ends of botholigonucleotides of the Cis-element decoy. The last six, five, four,three, two or the very last or one or more internucleotide linkageswithin the last six internucleotide linkages may be modified.Furthermore, different modifications of the internucleotide linkages maybe introduced into the nucleic acid. The double stranded DNAoligonucleotides generated in this way may be examined for theirsequence specific binding to IRF-1 by use of the routine EMSA assaysystem. This assay system permits the determination of the bindingconstant of the Cis-element decoy and thus the determination whether theaffinity has been changed by way of modification. Modified Cis-elementdecoys having still a sufficient binding may be selected wherein asufficient binding stands for at least about 50% or at least about 75%and more preferred about 100% of the binding of an unmodified nucleicacid.

Cis-element decoys with modified internucleotide linkages exhibit stilla sufficient binding may be examined whether they are more stable in thecell than the unmodified Cis-element decoys. Cells transfected withCis-element decoys according to the present invention are examined atdifferent times for the amount of the still existing Cis-element decoys.A Cis-element decoy labeled with a fluorescence dye (e.g. Texas red) orradioactively labeled (e.g. ³²P) is preferably used with a subsequentdigital fluorescence microscopy and autoradiography or scintigraphy,respectively. A successfully modified Cis-element decoy exhibits a halflife in the cell being higher than the half life of an unmodifiedCis-element decoy, preferably of at least about 48 hours, more preferredof at least about four days, most preferred of about at least aboutseven days.

Suitable modified internucleotide linkages are summarized in Uhlmann andPeyman ((1990) Chem. Rev. 90, 544). Modified internucleotide phosphatemoieties and/or non phosphorus bridges in a nucleic acid used in amethod of the present invention contain e.g. methyl phosphonate,phosphorothioate, phosphorodithioate, phosphoamidate, phosphate ester,whereas non phosphorus internucleotide analogues contain e.g. siloxanebridges, carbonate bridges, carboxymethylester bridges, acetamidatebridges and/or thioether bridges.

A further embodiment of the invention refers to the stabilization ofnucleic acids by introduction of structural features into the nucleicacid which increase the half life of the nucleic acid. Such structurescontaining the hairpin and bell like DNA are disclosed in U.S. Pat. No.5,683,985. Modified internucleotide phosphate moieties and/or nonphosphorus bridges may be simultaneously introduced together with theabove structures. The so generated nucleic acids may be examined withthe above described assay system for the binding and stability.

The core binding sequence may not only be present in a Cis-element decoybut also in a vector. In a preferred embodiment the vector is a plasmidvector and more preferred a plasmid vector capable to replicate in anautosomal fashion thereby increasing the stability of the introduceddouble stranded nucleic acid.

A further aspect of the present invention refers to a double strandedDNA oligonucleotide capable of binding to the transcription factor IRF-1in a sequence specific manner and having preferably one of the followingsequences whereby only one strand of the double stranded DNAoligonucleotide is shown below but the complementary strand is alsocomprised.

5′-SAAAnnnSAAAyy-3′, (SEQ ID NO:1) 5′-CAAAAGCGAAACC-3′, (SEQ ID NO:3)5′-GAAAAGCGAAACC-3′, (SEQ ID NO:5) 5′-CAAAAGTGAAACC-3′, (SEQ ID NO:7)5′-GAAAAGTGAAACC-3′, (SEQ ID NO:9) 5′-CAGAAAAGTGAAACCCTG-3′, (SEQ IDNO:11) 5′-CAGTTTCAAATTGAAACTG-3′, (SEQ ID NO:13)5′-CAGGAAAAGTGAAACCGCTG-3′, (SEQ ID NO:15) 5′-GCAGTTTCAAATTGAAAGTGC-3′,(SEQ ID NO:17) 5′-GGAAGCGAAAATGAAATTGACT-3′, (SEQ ID NO:19)5′-GGCAGTTTCAAATTGAAACTGCC-3′,. (SEQ ID NO:21)

Double stranded DNA oligonucleotides of the present invention exhibit alength, modifications and potentially a repeat of the specific bindingsite as described in detail above. The optimal length of the Cis-elementdecoy is chosen to optimize the binding to IRF-1 and the uptake into thecell. Usually a double stranded DNA oligonucleotide being shorter than12 bp binds only in a weak manner to its target protein whereas a doublestranded DNA oligonucleotide being longer than 22 bp is taken up by thecell only with low efficiency although it binds in a strong manner. Thebinding strength may be determined by EMSA whereas the uptake of thedouble stranded nucleic acid may be analyzed by means of a Cis-elementdecoy labeled with a fluorescent dye (e.g. Texas red) and radioactivelylabeled (e.g. ³²P) Cis-element decoy with subsequent digital fluorescentmicroscopy and autoradiography or scintigraphy, respectively. ACis-element decoy of the present invention may be stabilized asdescribed above.

A preferred embodiment of the present invention refers to Cis-elementdecoys containing a palindromic binding site and therefore comprising ina short double stranded nucleic acid at least two transcription factorbinding sites. The palindromic sequence does not necessarily entail ahigher binding of IRF-1 but said Cis-element decoy will be taken up morerapidly (more efficiently) by the target cells. However, particularlythe shorter Cis-element decoys according to the present invention arepalindromic only at the ends due to the long (centrally arranged) corebinding sequence and the repetitive G/CAAA motives. A preferably similarnumber of the single basis (A=C=G=T) may be used for a more efficientuptake, however, it is difficult to achieve a more efficient uptake dueto the repetitive G/CAAA motive of the Cis-element decoys according tothe present invention. A compromise is therefore preferred wherein atleast A=T and C=G. Further, preferably the core binding sequence may bearranged rather peripherally as being the case with some of thepreferred Cis-element decoy sequences.

A Cis-element decoy according to the present invention is quicklyincorporated into the cell. A sufficient uptake is characterized by themodulation of one or more genes which may be modulated by IRF-1. TheCis-element decoy according to the present invention modulatespreferably the transcription of one or more genes 4 hours after contactwith the cells, more preferred after about 2 hours, after about 1 hour,after about 30 min and most preferred after about 10 min. A mixtureusually used in such an experiment contains 10 μmol/L Cis-element decoy.

The present invention further relates to a method to modulate thetranscription of a least one gene in CD40 expressing cells, particularlyin endothelium cells, monocytes, dendritic cells, B lymphocytes, smoothmuscle cells, ceratinocytes or fibroblasts, wherein the method comprisesa step of contacting said cells with a mixture, containing one or moredouble stranded nucleic acids capable of binding to the transcriptionfactor IRF-1 in a sequence specific manner. A preferred method refers tothe use in endothelium cells being part of a transplant. The method isusually used at a transplant in vivo or ex vivo prior to theimplantation.

The transplants may be treated prior to the implantation by use of themethod according to the present invention ex vivo or after implantationby use of the method in vivo. The treated transplant is in a preferredembodiment of (a small) intestine, heart, liver, lung, kidney andpancreas and a combination of several organs, respectively. Thetreatment of the organs, more precisely the perfusion/incubation oftheir blood vessels with the Cis-element decoys according to the presentinvention may occur ex vivo by rinsing the solution immediately prior tothe implantation. The organ may be stored simultaneously in a suitableconservation solution (refrigerated) (e.g. University of WisconsinSolution, Brettschneider HTK solution).

The mixture containing the Cis-element decoys of the present inventionis contacted with the target cells (e.g. endothelium cells, monocytes,dendritic cells, B lymphocytes, smooth muscle cells, ceratinocytes orfibroblasts). The goal of said contacting is the transfer of theCis-element decoys binding IRF-1 into the target cell (i.e. the CD40expressing cell). Therefore, nucleic acid modification and/or additivesor adjuvants which are known to increase the penetration of membranesmay be used according to the present invention (Uhlmann and Peyman(1990) Chem. Rev. 90, 544).

A mixture according to the present invention contains in a preferredembodiment only nucleic acid and buffer. A suitable concentration of theCis-element decoys is in the range of at least 0,1 to 100 μmol/L,preferably at 10 μmol/L wherein one or more suitable buffers are added.One example of such buffer is tyrode solution, containing 144.3 mmol/LNa⁺, 4.0 mmol/L K⁺, 138.6 mmol/L Cl⁻, 1.7 mmol/L Ca²⁺, 1.0 mmol/L Mg²⁺,0.4 mmol/L HPO₄ ⁻², 19.9 mmol/L HCO₃ ⁻, 10.0 mmol/L D-glucose.

In a further embodiment of the present invention the mixture contains inaddition at least one additive and/or adjuvant. Additives and/oradjuvants like lipide, cationic lipids, polymers, liposomes,nanoparticles, nucleic acid aptamers, peptides and proteins being boundto DNA, or synthetic peptide DNA molecules are intended to increase e.g.the incorporation of nucleic acids into the cell, to target the mixtureto only one subgroup of cells, to prevent the degradation of the nucleicacid in a cell, to facilitate the storage of the nucleic acid mixtureprior to its use. Examples for peptides and proteins are syntheticpeptide DNA molecules e.g. antibodies, antibody fragments, ligands,adhesion molecules, which all may be modified or unmodified.

Additives stabilizing the Cis-element decoys in the cell are e.g.nucleic acid condensing substances like cationic polymers, poly L-lysineor polyethyleneimine.

The mixture being used in a method of the present invention ispreferably applied locally by injection, catheter, suppository, aerosols(nose and mouth spray, respectively, inhalation), trocars, projectiles,pluronic gels, polymers, which release medicaments permanently, or anyother means, which permit local access. Also the ex vivo use of themixture used in a method of the present invention permits a localaccess.

The inhibition of the IRF-1 activity may, however, be inhibited not onlyon protein level in the above described method but may be effected prioror at the translation of the transcription factor protein. A furtheraspect of the present invention refers, thus, to the provision of aninhibitor of the IRF-1 expression as a therapeutic substance. Saidinhibitor is preferably a single stranded nucleic acid molecule, a socalled antisense oligonucleotide. Antisense oligonucleotides may inhibitthe synthesis of a target gene at three different levels, at thetranscription (prevention of the hnRNA synthesis), the processing(splicing) of the hnRNA to mRNA and the translation of the mRNA inprotein on the ribosomes. The method to inhibit the expression of genesby antisense oligonucleotides is state of the art and well known to theskilled person. The antisense oligonucleotide against IRF-1 used in themethod of the present invention exhibits preferably the sequence5′-CGAGTGATGGGCATGTTGGC-3′ (SEQ ID NO:23) and bridges the start codon.Further preferred sequences of antisense oligonucleotides are5′-GATTCGGCTGGTCGC-3′ (SEQ ID NO:24), 5′-TAATCCAGATGAGCCC-3′ (SEQ IDNO:25) and 5′-GGAGCGATTCGGCTGGT-3′ (SEQ ID NO:26). The antisenseoligonucleotide may be a single stranded DNA molecule, RNA molecule or aDNA/RNA-hybrid molecule. Further, the antisense oligonucleotide mayexhibit one or more modified internucleotide linkages, e.g. the abovedescribed sequences of the Cis-element decoy. With an antisenseoligonucleotide stabilized by phosphorothioate modified internucleotidelinkages it has be preferably to be considered, that between the basescytosine and guanine no phosphorothioate modified internucleotidelinkage is introduced because this results in an IFNγ like activationpreferably of immune competent cells (e.g. endothelium cells) and, thus,would foil the desired therapeutic effect at least in part.

A further aspect according to the present invention is an antisenseoligonucleotide specifically inhibiting the IRF-1 expression andpreferably having one of the following sequences:

5′-CGAGTGATGGGCATGTTGGC-3′, (SEQ ID NO:23) 5′-GATTCGGCTGGTCGC-3′, (SEQID NO:24) 5′-TAATCCAGATGAGCCC-3′, (SEQ ID NO:25)5′-GGAGCGATTCGGCTGGT-3′. (SEQ ID NO:26)

A further aspect of the present invention refers further to the use ofthe antisense oligonucleotides and/or double stranded DNA moleculesaccording to the present invention for the manufacture of a medicamentfor the prevention and/or therapy of cardiovascular complications likethe restenosis after percutan angioplasty or the stenosis of venousbypasses, the chronic (graft arteriosclerosis or vasculopathy) or acutetransplant rejection, the graft versus host disease (GVAD),immunological hypersensitivity reactions (allergies), particularlybronchial asthma and atopic dermatitis, chronic recurrent inflammationdiseases, particularly colitis ulcerosa and Morbus Crohn, psoriasis andsarcoidosis, as well as autoimmune diseases, particularly diabetesmellitus, multiple sclerosis, collagenosis (for example systemic Lupuserythematodes), rheumatoid arthritis and vasculotids. A particularadvantage of this therapeutic approach further consists of thesimultaneous reduction of the TH1- and TH2-cell-response to which theCD40/CD154 signalling pathway effects co-stimulatory. Thereby it couldnot lead to a disinhibition of the TH1 cell reaction (for examplepsoriasis) with attenuation of the TH2 cell reaction (e.g. atopicdermatitis) and vice versa, respectively.

The following examples and figures explain the invention but are notintended to limit the scope of the invention.

1. Cell Culture

Human endothelium cells were isolated from umbilical veins by treatingwith 1.6 U/ml dispase in hepes modified tyrode solution for 30 min by37° C. The cells were cultivated on 6 well tissue cultures coated withgelatine (2 mg/ml gelatine in 0.1 M HCl for 30 min at room temperature;RT) in 1.5 ml M199 medium containing 20% fetal calf serum, 50 U/mlpenicillin, 50 μg/ml streptomycin, 10 U/ml nystatin, 5 mM HEPES and 5 mMTES, 1 μg/ml heparin and 40 μg/ml endothelial growth factor. The cellswere identified by their typical paving stone morphology, positiveimmunostaining for the von Willebrandt-Factor (vWF) and fluorimetricdetection (FACS) of PECAM-1 (CD31) as well as negative immunostainingfor smooth muscle α-Actin (Krzesz at al. (1999) FEBS Lett. 453, 191).

2. RT-PCR Analysis

The endothelial total RNA was isolated with the Qiagen RNeasy Kit(Qiagen, Hilden, Germany) with a subsequent cDNA Synthesis with themaximum of 3 μg RNA and 200 U Superscript™ II reverse transcriptase(Gibco Life Technologies, Karlsruhe, Germany) in a total volume of 20 μlaccording to the manufacturer's instructions. To adjust the cDNA load 5μl (approximately 75 ng cDNA) of the resulting cDNA solution and theprimer pair (Gibco) for the elongation factor 1 (EF-1) PCR with 1 U TaqDNA polymerase (Gibco) was used in a total volume of 50 μl. EF-1 wasused as internal Standard for the PCR. The PCR products were separatedon 1.5% agarose gels containing 0.1% ethidiumbromide and the intensitiesof the bands were determined densitometrically with a CCD camera systemand the One Dscan Gel Analysis Software from Scanalytics (Billerica,Mass., USA) to adjust the volume of the cDNA in subsequent PCR analyses.

All PCR reactions were individually performed for each primer pair in aHybrid OmnE Thermocycler (AWG, Heidelberg, Germany). The individual PCRconditions for the cDNA of human endothelial umbilical veins were asfollows: CD40 (amplicon size 381 bp, 25 cycles, annealing temperature60° C., (forward primer) 5′-CAGAGTTCACTGAAACGGAATGCC-3′ (SEQ ID NO:27),(reverse primer) 5′-TGCCTGCCTGTTGCACAACC-3′(SEQ ID NO:28); E-Selectin(amplicon size 304 bp, 33 cycles, annealing temperature 60° C., (forwardprimer) 5′-AGCAAGGCATGATGTTAACC-3′ (SEQ ID NO:29), (reverse primer)5′-GCATTCCTCTCTTCCAGAGC-3′ (SEQ ID NO:30); IRF-1 (amplicon size 310 bp,29 cycles, annealing temperature 55° C., (forward primer)5′-TTCCCTCTTCCACTCGGAGT-3′ (SEQ ID NO:31), (reverse primer)5′-GATATCTGGCAGGGAGTTCA-3′ (SEQ ID NO:32); EF-1 (amplicon size 220 bp,22 cycles, annealing temperature 55° C., (forward primer)5′-TCTTAATCAGTGGTGGAAG-3′ (SEQ ID NO:33), (reverse primer)5′-TTTGGTCAAGTTGTTTCC-3′ (SEQ ID NO:34).

3. Electrophoretic Mobility Shift Analysis (EMSA)

The nuclear extracts and [³²P]-labeled double stranded consensusoligonucleotides (Santa Cruz Biotechnologie, Heidelberg, Germany) nondenaturing polyacrylamidgelelectrophoreses, autoradiographie andsupershift analysis were performed as described by Krzesz at al (1999)FEBS Lett. 453, 191. Oligonucleotides were used with the followingsingle stranded sequences (core binding sequences are underlined): NFκB,5′-AGTTGAGGGGACTTTCCCAGGC-3′ (SEQ ID NO:35); STAT-1,5′-CATGTTATGCATATTCCTGTAAGT G-3′ (SEQ ID NO:36); IRF-1,5′-GGAAGCGAAAATGAAATTGACT-3′ (SEQ ID NO:19).

4. Decoy Oligonucleotide (dODN) Technique

Double stranded dODNs were generated from the complementary singlestranded phosphorothioate linked oligonucleotides (Eurogentec, Cologne,Germany) as described by Krzesz et al. (1999) FEBS Lett. 453, 191. Thecultivated human endothelium cells were pre-incubated for 4 hours with aconcentration of 10 μM of the respective dODN. These were the sameconditions which have been optimized previously due to EMSA and RT-PCRanalyses. The dODN containing medium was usually replaced afterwardswith fresh medium. The single stranded sequences of the dODNs are setforth below (underlined letters designate phosphorothioate linked bases,all sequences are written in 5′-3′ direction):

NFκB, AGTTGAGGGGACTTTCCCAGGC; (SEQ ID NO:35) STAT-1,CATGTTATGCATATTCCTGTAAGTG; (SEQ ID NO:36) IRF-1, GGAAGCGAAAATGAAATTGACT;(SEQ ID NO:19) IRF-1n CAGAAAAGTGAAACCCTG; (SEQ ID NO:11) cons IRF-1nCAGATGAGTGTAACCCTG. (SEQ ID NO:37) mut

5. Antisense Oligonucleotide Technique

3% Lipofectin (v/v) (Gibco Life Technologies, Karlsruhe, Germany) wasadded to 1 ml culture medium for an antisense experiment and wasincubated for 30 min at room temperature. The respective antisenseoligonucleotide (Eurogentec, Cologne, Germany) was subsequently added ina final concentration of 0.2 μM and incubated for further 15 min at roomtemperature. At the beginning of the experiment the respective amountsof Heparin and endothelial growth factor were added and the conventionalcell culture medium of the endothelium cell culture was replaced byantisense Lipofectin Medium. The antisense Lipofectin Medium was removedafter 5 hours and replaced by fresh cell culture medium. The sequence ofthe IRF-1 antisense oligonucleotide (IRF-1 AS) was5′-CGAGTGATGGGCATGTTGGC-3′(SEQ ID NO:23). A missense oligonucleotide(IRF-1 MS, 5′-CGAGTGGTAGACGTATTGGC-3′ (SEQ ID NO: 38)) and a scrambledoligonucleotide (IRF-1 SCR, 5′-GAGCTGCTGAGGTCGTTGAG-3′ (SEQ ID NO:39))were used as control oligos.

6. Fluorescence Activated Cell Sorting (FACS)

The endothelium cells to be analyzed were washed initially three timeswith 1 ml FACS buffer (PBS, 2% fetal calf serum sterile filtrated) eachresuspended subsequently in 2 ml FACS buffer. The fluorescence labeledantibody (Pharmingen, San Diego, USA) was added according to theinstructions of the manufacturer (20 μl/10⁶ cells) after centrifugation(300×g, 5 min, +4° C.) and determination of the total cell number(Neubauer Counting Chamber) and incubated for 30 min at +4° C. in thedark. The sample was subsequently washed with 2 ml FACS buffer andcentrifuged for 10 min at 300×g and +4° C. The supernatant was removedand the cell pellet was resuspended in 1 ml Cell Fix (PBS, 1%formaldehyde) and stored in the dark until measuring at +4° C. (EPICS®XLMCL, Coulter, Krefeld, Germany). The following antibodies were used:CD40, R-Phycoerythrin (RPE)- and Fluorescein Isothiocyanate(FITC)-conjugated; PECAM-1 (CD31), Fluorescein Isothiocyanate(FITC)-conjugated. The respective RPE- and FITC-conjugated isotypecontrols were used to determine unspecific cell antibody bindings.

7. Western Blot Analysis

The endothelium cells were macerated by 5 consecutive freeze thaw cyclesin liquid nitrogen and 37° C. (heating block, Kleinfelden, Germany).Protein extracts were generated as described by Hecker et al. (1994)Biochem J 299, 247. 20-30 μg Protein were separated with a 10%polyacrylamidegelelectrophoresis under denaturing conditions in thepresence of SDS according to a standard protocol and transferred to aBioTrace™ Polyvinylidene Fluoride Transfermembran (Pall Corporation,Roβdorf, Germany). A polyclonal primary antibody directed against theC-terminus of the CD40 protein (Research Diagnostics Inc., Flanders,N.J., USA) was used to detect the CD40 protein. The protein bands weredetected after adding a peroxidase coupled anti rabbit IgG (1:3000,Sigma, Deisenhofen, Germany) by means of a chemiluminescent method(SuperSignal Chemiluminescent Substrat; Pierce Chemical, Rockford, Ill.,USA) coupled with a subsequent autoradiography (Hyperfilm™ MP, AmershamPharmacia Biotech, Buckinghamshire, GB). The loading and transfer ofidentical protein amounts was shown by staining of the blot with blueink.

8. Statistical Analysis

Unless shown differently all data in figures and in the text are shownas mean±SD of n experiments. The statistical analysis was performed withthe Students t-Test for unpaired data with a p-value <0.05, which wastaken as statistically significant.

9. Experimental Proof on Animals of the CD40/CD154 Associated TransplantRejection

The transplant rejection in rats was examined experimentally on animalsby use of a STAT-1 decoy oligonucleotide because STAT-1 is responsiblein rats for the Interferon-γ induced CD40 expression rather than ofIRF-1 as in humans (Krzesz et al. (1999) FEBS Lett. 453, 191).

Strain Combination

The strain combination Brown Norway donor was used for the allogenictransplantation to Lewis recipients. Without an immunosuppression thetransplant was rejected after 7 days. The transplantation from Lewis toLewis war performed as syngenic control.

Explantation

The abdomen of an animal was opened in the mid line under etherinhalation narcosis. An aorta segment was first released from allarterial parts so that approximately a 1 cm long aortic segment withprospective Ateria mesenterica was prepared. The complete colon wasremoved in the next step. Thereafter all venous vessels of the portalvein were ligated in the latitude of the pancreas so that the pancreaswas unrestrained into the porta hepatis. The so prepared donor smallintestine was now attached only to the trunk of the aorta and the portalvein. The aorta was clamped proximal and distal of the ateriamesenterica, the portal vein was severed at the porta hepatis and thevascular bed of the small intestine was rinsed with cold University ofWisconsin (UW) solution until no macroscopic blood residues were left inthe vascular bed. The intestine lumen was likewise rinsed in the laststep with cold UW solution, the intestine with an aorta segment wastaken off and stored in cold UW solution until implantation occurred (upto 120 min). When the transplant was treated with the STAT-1 DecoyOligonucleotide (sequence: CATGTTATGCATATTCCTGTAAGTG; (SEQ ID NO:36)) orthe respective mutated control nucleotide (sequence:CATGTTATGCAGACCGTAGTAAGTG (SEQ ID NO:40)), the transplant was infusedinto the arteria mesenterica in Ringer solution (containing 145 mmol/LNa⁺, 5 mmol/L K⁺, 156 mmol/L Cl⁻, 2 mmol/L Ca²⁺, 1 mmol/L Mg²⁺, 10mmol/L Hepes, 10 mmol/L D-glucose, pH 7.4; volume 3 ml, finalconcentration 20 μmol/L) and rinsed with Ringer solution immediatelybefore the anastomorization.

Implantation

The abdomen of an animal was opened in the mid line in ether inhalationnarcosis. The aorta and vena cava were prepared and clampedsimultaneously. Vessel connection was done in end-to-side in ancontinuing suture technique with a 8-0 nylonstitch. The ateriamesenterica carrying the aorta segment was anastomosed at the infravenalaorta and the portal vein was anastomosed at the infravenal vena cava.After release of the circulation the terminal ileum of the donorintestine was connected end-to-side to the terminal ileum of the donorintestine also by a 6-0 nylonstitch. The mucus produced by the donorintestine was drained off into the normal passage of the animal. Theoral end of the donor intestine was closed by ligature and the abdomenwas continuously disclosed in a two layer fashion. The animals receivepostoperatively for analgesia reasons Temgesic into their drinkingwater.

Intravital Microscopic

An assessment of the importance of the leucocyte endothelium interactionfor the inflammation reaction was possible only by intravital microscopyanalysis. This method enabled an observation of the “rolling andadhering” of leucocytes at the endothelium in vivo as well as anquantitative analysis of microvascular parameters (perfusion of thetissue, functional capillary density and blood flow).

The intravital microscopy was performed with a Axiotech Vario 100microscope from Zeiss (Göttingen) endowed with a HBO 100 mercury lampfor epifluorescence measurements. With the 10×, 20× and 40× (waterimmersions) lenses a solution of 243×, 476× and 933× was achieved. Themicroscopic images were taken with a CCD video camera (CF 8/1, Kappa)and stored for analysis on a video tape.

The rats (6 animals per group) were examined 7 days after thetransplantation in deep diethylether narcosis with intravitalmicroscopy. To facilitate the breathing the trachea was cannulated. Apolyurethane catheter was positioned into the arteria carotis forpermanent monitoring of the blood pressure for the simplification of theapplication of dyes. The body temperature of the animals was heldconstant with a heatable plate. The animals were opened by a ventralmedian cut, the colon descendens was evacuated, a small cut was set antimesenterially and the intestine was fixed in a specific fixture tofacilitate the microscopy. To prevent a drying of the tissue, theintestine was moistened permanently with Ringer solution. The intestinemicrocirculation was made visible by the injection of 0.8 ml 0.5% FITC(fluorescein isothiocyanate) coupled dextran. To cover the measurementsstatistically a least ten different areas of the respective intestinepart was examined. The different parameters were quantified as follows:The perfusion index resulted from the perfused mucosa areas (in %)+0.5×of all irregularly perfused mucosa areas (in %). The functionalcapillary density was determined by a computer aided image analysis(CAP-IMAGE software, Zeintl, Heidelberg, Germany). To examine theleukocyte endothelial interactions the leukocytes were labeled by theinjection of 0.2 ml 0.1% Rhodamine-6 G (Sigma, Heidelberg, Germany) andthe post capillary venoles were microscoped in the submucosa. Thoseleukocytes were defined as adherent leukocytes (“sticker”) whichattached to a vessel segment 100 μm length for at least 20 sec at theendothelium. The number of the sticker number/mm² endothelium surfacewas calculated. The endothelium surface resulted from the surfacecalculation for a cylinder.

Results of the Small Intestine Transplantation

The mucosal functional capillary density as a unit of measurement of theperfusion, was reduced down to 10% of the values of syngenictransplanted small intestines both in the control group and in the grouptreated with the mutated control oligonucleotides without rejection. Thefunctional capillary density was increased four times in contrast insmall intestines treated with the STAT-1 Cis-element decoy. The bloodflow (flow rate of the erythrocytes) was in these animals 10 timeshigher and the perfusion index was 3 times higher. The staseindex wasreduced for 60% and the number of the leucocytes attached to theendothelium was reduced for 25%. Only the latter parameter was notstatistically significant altered. The rejection induced reduction ofthe intestine perfusion and thus, the degeneration of the transplant wasin summary significantly reduced in the group treated with theCis-element decoy.

1. An inhibitor of the IRF-1 expression and/or activity as therapeuticsubstance.
 2. The inhibitor according to claim 1, wherein the inhibitoris a double stranded DNA molecule and inhibits the IRF-1 activity. 3.The inhibitor according to claim 2 having a nucleic acid sequenceaccording to SEQ ID NO: 1 to
 22. 4. The inhibitor according to claim 2,wherein the double stranded DNA molecule exhibits modifiedinternucleotide linkages.
 5. The inhibitor according to claim 1, whereinthe inhibitor is an antisense oligonucleotide and inhibits the IRF-1expression.
 6. The inhibitor according to claim 5 having a nucleic acidsequence according to SEQ ID NO:23 to
 26. 7. The inhibitor according toclaim 5, wherein the antisense oligonucleotide exhibits modifiedinternucleotide linkages.
 8. A method for the prevention or therapy ofcardiovascular complications chronic (graft atherosclerosis orvasculopathy) or acute transplant rejection, graft versus host disease(GVAD), immunological hypersensitivity reactions (allergies), chronicrecurrent inflammation, psoriasis and sarcoidosis, disease or autoimmunedisease, comprising administering to a subject in need thereof aninhibitor of IRF-1.
 9. An antisense oligonucleotide having a nucleicacid sequence according to SEQ ID NO:23 to
 26. 10. The antisenseoligonucleotide according to claim 9, wherein the antisenseoligonucleotide exhibits modified internucleotide linkages.
 11. Adouble-stranded DNA molecule having a nucleic acid sequence according toSEQ ID NO:1 to
 21. 12. The double-stranded DNA molecule according to 11,wherein the double stranded DNA molecule exhibits modifiedinternucleotide linkages.
 13. The method of claim 8, wherein saidcardiovascular complication is selected from the group consisting ofrestenosis after percutaneous angioplasty or the stenosis of venousbypasses.
 14. The method of claim 8, wherein the immunologicalhypersensitivity reaction is selected from the group consisting ofbronchial asthma and atopic dermatitis
 15. The method of claim 8,wherein the chronic recurrent inflammation diseases is selected from thegroup consisting of colitis ulcerosa and Morbus Crohn.
 16. The method ofclaim 8, wherein the autoimmune disease is selected from the groupconsisting of diabetes mellitus, multiple sclerosis, collagenosis (forexample systemic Lupus erythematodes), rheumatoid arthritis andvasculotids.